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Poster presented at the Eight International Symposium on Molecular Insect Science, held 7-10th July 2019 in Barcelona (Spain)., The Piwi-interacting RNAs (piRNAs) are a small non-coding RNAs involved in the maintenance of genome stability. piRNAs were described as expressed exclusively in germinal cells, but their presence in somatic cells has been taking relevance in the last years. We have identified the Blattella germanica piRNAs from 24 small RNA libraries, corresponding to different developmental stages (Llonga et al 2018 DOI: 10.1002/jez.b.22815). Now we focus on the identification of piRNAs from a maternal origin and the characterization of the piRNAs involved in the transition to the zygotic stage. The role of such piRNAs in the protection of the embryo genome and the relationship with their putative target RNAs (transposable elements and mRNAs) are key to the understanding of the general functions of these regulatory elements but also their particularities in hemimetabolous insects. The 24 small RNA libraries were aligned against the genome with Bowtie and R Statistical Software was used for expression analysis. Finally, ovary and early embryonic piRNA expressions were validated by qRT-PCR. The analysis of B. germanica individual small RNA libraries, corresponding to ovaries from 7-day-old adult females (OvD7), non-fertilized eggs (NFE), the zygote (ED0), and the first day of embryogenesis (ED1), show that piRNAs in these stages agree with the current model of two piRNA biogenesis pathways. The most expressed piRNAs in each library of this transition have been analyzed in order to identify specific functions for each stage. By mapping the sequence to the genome, we have identified the origin of these piRNAs and also a number of potential mRNAs targets.

Poster presented at the Eight International Symposium on Molecular Insect Science, held 7-10th July 2019 in Barcelona (Spain)., Oogenesis, the formation and maturation of an oocyte, is a crucial process in the female reproductive physiology in animals. It encompasses a series of events that must occur in the correct order and at the right time. Hormonal signals are fundamental in the oogenesis processes, for oocyte differentiation and growth, to determine changes in cell program or for chorion synthesis. However, the maintenance of genome stability in insect germ cells during the proliferation and differentiation is of paramount importance, and it is the Piwi pathway which plays this role. Using Blattella germanica as a model, a species representative of panoistic ovaries, we studied three of the core proteins of the Piwi pathway in the ovaries, in order to illuminate their function in the maintenance of germ cells stability. B. germanica transcriptomes and bioinformatic analyses were used to identify the main proteins involved in the biogenesis of piRNAs and its regulation. By quantitative real-time PCR, the expression of Piwi, Argonaute 3 (Ago3) and Yb (fs(1)Yb or Tudor 12) was analyzed in B. germanica ovaries, and their functions were studied by RNAi. Piwi, Ago3 and Yb, are mainly expressed in ovaries of last nymphal instar when the differentiation of germinal cells is usually arrested. The expression of these genes is maintained during the first gonadotrophic cycle. Depletion of Yb and Ago3 allowed us to demonstrate their involvement in the primary and secondary piRNAs pathway, respectively.

Poster presented at the Eight International Symposium on Molecular Insect Science, held 7-10th July 2019 in Barcelona (Spain)., Interference of RNA (RNAi) is a cellular process that allows the cell to protect itself against harmful agents, especially alien RNA. Through the treatment with double-stranded RNA (dsRNA), this mechanism is being used to perform specific knockdown of the expression of a given gene, in order to do laboratory functional genetic studies or even to control insect pests. The aim of the present work is to study an aspect of the functioning of RNAi in insects, in particular, to determine the functions the protein Argonaute-2 (Ago2). Using the cockroach Blattella germanica as a model, specimens treated with dsRNA against Ago2 (dsAgo2), and control, were subsequently treated with a heterologous dsRNA (dsPolyh). Four days after the second treatment, libraries of small RNAs were obtained from control and dsAgo2 depleted specimens. The small interfering RNAs (siRNAs) corresponding to dsPolyh were analyzed. siRNAs found in the libraries were not equally distributed along the whole length of both RNA strands from the dsRNA injected, but enriched in certain areas. This unequal distribution was similar both in Control- and dsAgo2-treated specimens. Nevertheless, dsAgo2 samples contained 4 times more 22 nt siRNAs (the most usual size in the libraries) than Control samples. The unequal distribution of siRNAs along a heterologous dsRNA sequence indicates that there is a selection of siRNAs independent of their use in the process of degradation of the target RNA. The similar distribution of siRNAs in control and dsAgo2 samples indicates that Ago2 is not the protein responsible for doing siRNA selection. Nevertheless, the higher amount of 22 nt siRNAs in dsAgo2-treated specimens shows Ago2 nuclease activity on ¿mature¿ siRNAs.

A high consumption of fat and simple sugars, especially fructose, has been related to the development of insulin resistance, but the mechanisms involved in the effects of these nutrients are not fully understood. This study investigates the effects of a Western-type diet and liquid fructose supplementation, alone and combined, on insulin signalling and inflammation in low-density lipoprotein (LDL) receptor-deficient mice (LDL-R−/−). LDL-R−/− mice were fed chow or Western diet ±15% fructose solution for 12 weeks. Plasma glucose and insulin, and the expression of genes related to inflammation in the liver and visceral white adipose tissue (vWAT), were analysed. V-akt murine thymoma viral oncogene homolog-2 (Akt) activation was measured in the liver of the mice after a single injection of saline or insulin. None of the dietary interventions caused inflammation in vWAT, whereas the Western diet induced hepatic inflammation, which was further enhanced by liquid fructose, leading also to a significant increase in fibrogenesis markers. However, there was no change in plasma glucose or insulin, or insulin-induced Akt phosphorylation. In conclusion, hepatic inflammation and fibrogenesis markers induced by a Western diet supplemented with liquid fructose in LDL-R−/− mice are not associated with a significant impairment of hepatic insulin signalling.

The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies higher than 20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make about 80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model. This work benefited from preliminary Nanopore RNA-seq data analyses performed by Bea Calvo and Audald Lloret-Villas, as well as discussions with Eduardo Eyras and Ivan de la Rubia. We acknowledge funding from Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) grant PGC2018–094091-B-I00, cofunded by Fondo Europeo de Desarrollo Regional (FEDER), and from Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, grant 2017SGR01020.